Cysteine-scanning analysis of the nucleobase-ascorbate transporter signature motif in YgfO permease of Escherichia coli: Gln-324 and Asn-325 are essential, and Ile-329-Val-339 form an alpha-helix

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dc.contributor.authorKaratza, P.en
dc.contributor.authorPanos, P.en
dc.contributor.authorGeorgopoulou, E.en
dc.contributor.authorFrillingos, S.en
dc.date.accessioned2015-11-24T19:25:38Z
dc.date.available2015-11-24T19:25:38Z
dc.identifier.issn0021-9258-
dc.identifier.urihttps://olympias.lib.uoi.gr/jspui/handle/123456789/22642
dc.rightsDefault Licence-
dc.subjectAmino Acid Motifs/geneticsen
dc.subjectAmino Acid Sequenceen
dc.subjectAmino Acid Substitution/*geneticsen
dc.subjectAsparagine/geneticsen
dc.subjectBiological Transport, Active/geneticsen
dc.subjectCysteine/chemistry/*geneticsen
dc.subjectEscherichia coli Proteins/*chemistry/*genetics/metabolismen
dc.subjectGlutamine/geneticsen
dc.subjectIsoleucine/geneticsen
dc.subjectMolecular Sequence Dataen
dc.subjectMutagenesis, Site-Directeden
dc.subjectNucleobase Transport Proteins/*chemistry/*genetics/metabolismen
dc.subjectProtein Structure, Secondary/geneticsen
dc.subjectValine/geneticsen
dc.subjectXanthine/metabolismen
dc.titleCysteine-scanning analysis of the nucleobase-ascorbate transporter signature motif in YgfO permease of Escherichia coli: Gln-324 and Asn-325 are essential, and Ile-329-Val-339 form an alpha-helixen
heal.abstractThe nucleobase-ascorbate transporter (NAT) signature motif is a conserved sequence motif of the ubiquitous NAT/NCS2 family implicated in defining the function and selectivity of purine translocation pathway in the major fungal homolog UapA. To analyze the role of NAT motif more systematically, we employed Cys-scanning mutagenesis of the Escherichia coli xanthine-specific homolog YgfO. Using a functional mutant devoid of Cys residues (C-less), each amino acid residue in sequence (315)GSIPITTFAQNNGVIQMTGVASRYVG(340) (motif underlined) was replaced individually with Cys. Of the 26 single-Cys mutants, 16 accumulate xanthine to > or =50% of the steady state observed with C-less YgfO, 4 accumulate to low levels (10-25% of C-less), F322C, N325C, and N326C accumulate marginally (5-8% of C-less), and P318C, Q324C, and G340C are inactive. When transferred to wild type, F322C(wt) and N326C(wt) are highly active, but P318G(wt), Q324C(wt), N325C(wt), and G340C(wt) are inactive, and G340A(wt) displays low activity. Immunoblot analysis shows that replacements at Pro-318 or Gly-340 are associated with low or negligible expression in the membrane. More extensive mutagenesis reveals that Gln-324 is critical for high affinity uptake and ligand recognition, and Asn-325 is irreplaceable for active xanthine transport, whereas Thr-332 and Gly-333 are important determinants of ligand specificity. All single-Cys mutants react with N-ethylmaleimide, but regarding sensitivity to inactivation, they fall to three regions; positions 315-322 are insensitive to N-ethylmaleimide, with IC(50) values > or =0.4 mM, positions 323-329 are highly sensitive, with IC(50) values of 15-80 microM, and sensitivity of positions 330-340 follows a periodicity, with mutants sensitive to inactivation clustering on one face of an alpha-helix.en
heal.accesscampus-
heal.fullTextAvailabilityTRUE-
heal.identifier.primary10.1074/jbc.M605748200-
heal.identifier.secondaryhttp://www.ncbi.nlm.nih.gov/pubmed/17077086-
heal.identifier.secondaryhttp://www.jbc.org/content/281/52/39881.full.pdf-
heal.journalNameJ Biol Chemen
heal.journalTypepeer-reviewed-
heal.languageen-
heal.publicationDate2006-
heal.recordProviderΠανεπιστήμιο Ιωαννίνων. Σχολή Επιστημών Υγείας. Τμήμα Ιατρικήςel
heal.typejournalArticle-
heal.type.elΆρθρο Περιοδικούel
heal.type.enJournal articleen

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